The field of the invention is protein kinase enzymes involved in glycogen metabolism, in signal transduction, and in cellular regulation of enzyme activity and transcription.
Lithium is an effective drug for the treatment of bipolar (manic-depressive) disorder (Price et al., 1994, New Eng. J. Med. 331:591-598; Goodwin et al., 1990, In: Manic-Depressive Illness, New York: Oxford University Press). Lithium is not only effective for treatment of acute episodes of mania, but this compound also reduces the frequency and severity of recurrent episodes of mania and depression in patients with bipolar and unipolar disorders (Goodwin, et al., 1990, supra). Lithium can be used to treat profound depression in some cases. Despite the remarkable efficacy of lithium observed during decades of its use, the molecular mechanism(s) underlying its therapeutic actions have not been fully elucidated (Bunney, et al., 1987, In: Psychopharmacology: The Third Generation of Progress, Hy, ed., New York, Raven Press, 553-565; Jope et al., 1994, Biochem. Pharmacol. 47:429-441; Risby et al., 1991, Arch. Gen. Psychiatry 48:513-524; Wood et al., 1987, Psychol. Med. 17:570-600).
Lithium does not have an immediate effect during the treatment of mania, but rather requires several weeks to manifest a clinical response. It has been suggested that this delay reflects changes in the expression of genes involved in alleviation of mania (Manji et al., 1995, Arch. Gen. Psychiatry 52:531-543).
In addition to its use as a therapeutic drug for the treatment of mania, lithium exhibits numerous physiological effects in animals. For example, lithium mimics insulin action by stimulating glycogen synthesis (Bosch et al., 1986, J. Biol. Chem. 261:16927-16931). Further, exposure to lithium has dramatic morphogenic effects during the early development of numerous organisms. The effects of lithium on the development of diverse organisms, including Dictyostelium, sea urchins, zebrafish, and Xenopus have been reported (Maeda, 1970, Dev. Growth and Differ. 12:217-227; Van Lookeren Campagne et al., 1988, Dev. Genet. 9:589-596; Kao et al., 1986, Nature 322:371-373; Stachel et al., 1993, Development 117:1261-1274; Livingston et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3669-3673). In Dictyostelium discoideum, lithium alters cell fate by blocking spore cell development and promoting stalk cell development (Maeda, 1970, supra; Van Lookeren Campagne et al., 1988, supra). In Xenopus, lithium induces an expansion of dorsal mesoderm, leading to duplication of the dorsal axis or, in extreme cases, entirely dorsalized embryos which lack identifiably ventral tissues (Kao et al., 1986, Nature 322:371-373). Lithium also rescues UV-ventralized embryos (Kao et al., 1986, supra). In addition, treatment of sea urchin animal blastomeres with lithium induces the blastomeres to display a morphology resembling that of isolated vegetal blastomeres (Horstadius, 1973, In: Experimental Embryology of Echinoderms, Oxford University Press, Oxford).
Even though lithium is remarkably effective for the treatment of mania in many human patients, lithium treatment in humans is accompanied by several serious drawbacks (Baraban, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5738-5739). Particularly troublesome is the slim margin between therapeutic and toxic levels of lithium in vivo. Furthermore, because clearance of lithium is intimately tied to sodium and water excretion, a slight change in electrolyte balance can precipitate a life-threatening increase in lithium levels in vivo (Baraban, supra). In addition, even tight regulation of lithium within its therapeutic window is associated with a wide range of side effects, such as tremor, renal dysfunction, thyroid abnormalities, and birth defects (Jefferson et al., 1989, In: Comprehensive Textbook of Psychiatry, Kaplan et al., eds., Williams and Wilkins, Baltimore, vol. 2, 1655-1662). It is recommended that facilities for prompt and accurate serum lithium determinations be available before administering lithium to a patient (Physicians Desk Reference, 51 st Ed., 1997, p. 2658). In addition, lithium should generally not be administered to patients having significant renal or cardiovascular disease, severe debilitation or dehydration, sodium depletion, or to patients receiving diuretics, since the risk of lithium toxicity is very high in such patients (Physicians Desk Reference, 1997, supra, at 2352). Numerous other side effects are detailed in the Physicians Desk Reference (1997, supra, at 2352, 2658).
The mechanism or mechanisms by which lithium exerts these diverse effects are unclear (Price et al., 1994, New Eng. J. Med. 331:591-598; Goodwin et al., 1990, In: Manic-Depressive Illness, New York, Oxford University Press; Berridge et al., 1989, Cell 59:411-419; Avissar et al., 1988, Nature 331:440-442). A favored hypothesis, the inositol depletion hypothesis, is based on the observation that lithium inhibits inositol monophosphatase (IMPase) and, by doing so, depletes cells of endogenous inositol (Berridge et al., 1989, Cell 59:411-419; Hallcher et al., 1980, J. Biol. Chem. 255:10896-10901). Cells that do not have an exogenous source of inositol would, in principle, be unable to synthesize phosphatidyl-3-inositol phosphate, the precursor of inositol 1,4,5 tris-phosphate (IP3). Thus, according to the inositol depletion hypothesis, lithium-treated cells are unable to generate IP3 in response to extracellular signals and, as a consequence, IP3-dependent responses are blocked. Some experimental results appear to support the inositol depletion hypothesis (Baraban, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5738-5739; Berridge et al., 1989, Cell 59:411-419; Manji et al., 1995, Arch. Gen. Psychiatry 52:531-543; Busa et al., 1989, Dev. Biol. 132:315-324). However, other experimental results do not support this hypothesis (Klein et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93:8455-8459; Drayer et al., 1994, EMBO J. 13:1601-1609).
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase having a 47 kDa monomeric structure. It is one of several protein kinases which phosphorylates glycogen synthase (Embi, et al., 1980, Eur. J. Biochem., 107:519-527; Hemmings et al., 1982, Eur. J. Biochem. 119:443-451). GSK-3 is also referred to in the literature as factor A (FA) in the context of its ability to phosphorylate FC, a protein phosphatase (Vandenheede et al., 1980, J. Biol. Chem. 255:11768-11774). Other names for GSK-3 and homologs thereof include zeste-white3/shaggy (zw3/sgg; the Drosophila melanogaster homolog), ATP-citrate lyase kinase (ACLK or MFPK; Ramakrishna et al., 1989, Biochem. 28:856-860; Ramakrishna et al., 1985, J. Biol. Chem. 260:12280-12286), GSLA (the Dictyostelium homolog; Harwood et al., 1995, Cell 80:139-48), and MDSI, MCK1, and others (yeast homologs; Hunter et al., 1997, TIBS 22:18-22).
The gene encoding GSK-3 is highly conserved across diverse phyla. GSK-3 exists in two isoforms in vertebrates, GSK-3xcex1 and GSK-3xcex2. In vertebrates, the amino acid identity among homologs is in excess of 98% within the catalytic domain of GSK-3 (Plyte et al., 1992, Biochim. Biophys. Acta 1114:147-162). It has been reported that there is only one form of GSK-3 in invertebrates, which appears to more closely resemble GSK-3xcex2 than GSK-3xcex1. Amino acid similarities (allowing for conservative replacements) between the slime mold and fission yeast proteins with the catalytic domain of human GSK-3xcex2 are 81% and 78%, respectively (Plyte et al., 1992, supra). The remarkably high degree of conservation across the phylogenetic spectrum suggests a fundamental role for GSK-3 in cellular processes.
GSK-3 has been demonstrated to phosphorylate numerous proteins in vitro, including, but not limited to glycogen synthase, phosphatase inhibitor I-2, the type-II subunit of cAMP-dependent protein kinase, the G-subunit of phosphatase-1, ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein, a microtubule-associated protein, a neurofilament protein, an N-CAM cell adhesion molecule, nerve growth factor receptor, c-Jun transcription factor, JunD transcription factor, c-Myb transcription factor, c-Myc transcription factor, L-myc transcription factor, adenomatous polyposis coli tumor suppressor protein, xcfx84 protein, and xcex2-catenin (Plyte et al., 1992, Biochim. Biophys. Acta 1114:147-162; Korinek et al., 1997, Science 275:1784-1787; Miller et al., 1996, Genes and Dev. 10:2527-2539). The phosphorylation site recognized by GSK-3 has been determined in several of these proteins (Plyte et al., 1992, supra). The diversity of these proteins belies a wide role for GSK-3 in the control of cellular metabolism, growth, and development. GSK-3 tends to phosphorylate serine and threonine residues in a proline-rich environment, but does not display the absolute dependence upon these amino acids which is displayed by protein kinases which are members of the mitogen-activated protein (MAP) kinase or cdc2 families of kinase enzymes.
Among the proteins which are phosphorylated by GSK-3 is c-Jun, the expression product of the c-jun proto-oncogene and the cellular homolog of the v-jun oncogene of avian sarcoma virus (Dent et al., 1989, FEBS Lett. 248:67-72). Jun acts as a component of the activator protein-1 (AP-1) transcription factor complex, which binds to a palindromic consensus binding site (the AP-1 site). c-Jun is both necessary and sufficient to induce transcription of genes having an AP-1 site (Angel et al., 1988, Nature 332:166-171; Angel et al., 1988, Cell: 55:875-885; Chiu et al., 1988, Cell 54:541-552; Bohmann et al., 1989, Cell 59:709-717; Abate et al., 1990, Mol. Cell. Biol. 10:5532-5535). Transcription of a gene having an AP-1 site may be initiated by either a Fos-Jun heterodimer or by a Jun-Jun homodimer, although the Fos-Jun heterodimer binds to DNA more stably than the Jun-Jun homodimer and is consequently a more potent transcription activator. Fos is the expression product of another proto-oncogene, c-fos (Schonthal et al., 1988, Cell 54:325-334; Sassone-Corsi, 1988, Nature 334:314-319). Phosphorylation of c-Jun by GSK-3 severely reduces the binding affinity of Jun-Jun homodimer for AP-1 sites (Boyle et al., 1991, Cell 64:573-584; Plyte et al., 1992, supra).
GSK-3 is a negative regulator of the wnt signaling pathway. The wnt pathway is a highly conserved signaling pathway that regulates cell fate decisions in both vertebrates and invertebrates (Perrimon, 1994, Cell 76:781-784; Perrimon, 1996, Cell 86:513-516; Miller et al., 1996, Genes and Dev. 10:2527-2539). Much of the pathway has been determined from detailed genetic analysis in Drosophila. At present, identified components of this signaling pathway include wnts (the secreted ligand), frizzled (the wnt receptor), and the intracellular mediators disheveled, GSK-3 (denoted zw3/sgg in Drosophila), and xcex2-catenin (denoted armadillo in Drosophila). In 10T1/2 cells, wnt signaling inhibits GSK-3 p enzymatic activity (Cook et al., 1996, EMBO J. 15:4526-4536). This result is consistent with epistasis experiments in Drosophila which suggest an inhibitory role for GSK-3xcex2/zw3/sgg in the wnt pathway. Wnt signaling leads to stabilization of xcex2-catenin protein in Drosophila (Peifer et al., 1994, Dev., 120:369-380; van Leeuwen, et al., 1994, Nature 368:342-344) as well as Xenopus (Yost et al., 1996, Genes and Dev., 10:1443-1454). It has also been demonstrated that treatment of Drosophila S2 cells with LiCl leads to accumulation of armadillo protein (Stambolic et al., 1996, Curr. Biol. 6:1664-1668). Stabilization of xcex2-catenin is associated with translocation of xcex2-catenin to the nuclei of cells responding to wnt signaling (Funayama et al., 1995, J. Cell Biol., 128:959-968; Schneider et al., 1996, Mech. Dev., 57:191-198; Yost et al., 1996, supra). In addition, ectopic expression of conserved genes, including wnts, disheveled, and xcex2-catenin, leads to second axis formation in Xenopus. Second axis formation in Xenopus is also observed following lithium treatment. Although xcex2-catenin was originally discovered as a cadherin-binding protein, it has recently been shown to function as a transcriptional activator when complexed with members of the Tcf family of DNA binding proteins (Molenaar et al., 1996, Cell 86:391; Behrens et al., 1996, Nature 382:638).
There exists a pressing need to identify compositions which have the therapeutic effect of lithium without the attendant side effects which accompany administration of lithium to human patients.
The invention relates to a method of identifying a GSK-3 inhibitor comprising providing a mixture comprising GSK-3, a source of phosphate, a GSK-3 substrate and a GSK-3 assay buffer, incubating the mixture in the presence or absence of a test compound, and measuring the level of phosphorylation of the GSK-3 substrate, wherein a lower level of phosphorylation of the GSK-3 substrate in the presence of the test compound compared with the level of phosphorylation of the GSK-3 substrate in the absence of the test compound is an indication that the test compound is a GSK-3 inhibitor.
The method of identifying a GSK-3 inhibitor may be performed either in vitro wherein the assay mixture is cell-free, in vitro wherein live cells are included in the assay, or in vivo in an animal.
The GSK-3 may be provided in the assay mixture as a protein or as a nucleic acid, either DNA or RNA, from which GSK-3 is expressed.
In one aspect of the invention, the mixture is contained within a eukaryotic cell.
In one embodiment of the invention, at least one of the GSK-3, the GSK-3 substrate and the test compound is injected into the eukaryotic cell prior to the incubation. In another embodiment, at least two of the GSK-3, the GSK-3 substrate and the test compound are injected into the eukaryotic cell prior to the incubation. In yet another embodiment, the GSK-3, the GSK-3 substrate and the test compound are injected into the eukaryotic cell prior to the incubation.
In another embodiment of the invention, the eukaryotic cell is suspended in a solution comprising the test compound.
The eukaryotic cell is selected from the group consisting of a Xenopus laevis oocyte, a Xenopus laevis embryo cell, a mammalian cell, a Drosophila melanogaster S2 cell, a Dictyostelium discoideum cell and a yeast cell. Preferably, the eukaryotic cell is selected from the group consisting of a Xenopus laevis oocyte and a Xenopus laevis embryo cell. More preferably, the eukaryotic cell is a Xenopus laevis oocyte which even more preferably, is aXenopus laevis embryo cell, yet more preferably, aXenopus laevis embryo ventral vegetal blastomere cell.
In one aspect of the invention, the phosphate source comprises a nucleotide triphosphate selected from the group consisting of ATP and GTP and preferably comprises a detectable label which is transferred to the substrate during the incubation. More preferably, the phosphate source comprises [xcex332P]-ATP.
The GSK-3 which is contained within the mixture may be endogenous in the eukaryotic cell.
Preferably, the GSK-3 is selected from the group consisting of human GSK-3xcex1, human GSK-3xcex2, Xenopus laevis GSK-3xcex1, Xenopus laevis GSK-3xcex2, bacterially-expressed Xenopus laevis GSK-3xcex2, bacterially-expressed rat GSK-3xcex2, the expression product of the Drosophila melanogaster zw3/sgg gene, and the expression product of the Dictyostelium discoideum gskA gene. More preferably, the GSK-3 is bacterially-expressed rat GSK-3xcex2.
The GSK-3 substrate which is contained within the mixture may also be endogenous in the eukaryotic cell.
Preferably, the GSK-3 substrate is selected from the group consisting of glycogen synthase, phosphatase inhibitor I-2, cAMP-dependent protein kinase type II subunit, phosphatase- 1 G-subunit, ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein, a microtubule-associated protein, a neurofilament protein, an N-CAM cell adhesion molecule, nerve growth factor receptor, c-Jun, JunD, c-Myb, c-Myc, L-myc, adenomatous polyposis coli tumor suppressor protein, xcfx84 protein, xcex2-catenin, peptide GS-2, and peptide derivatives of any of these which comprise a GSK-3 phosphorylation site. More preferably, the GSK-3 substrate comprises xcfx84 protein.
The test compound used in the method of the invention is selected from the group consisting of bis-indolyl maleimides, staurosporine and derivatives thereof, and protein kinase C inhibitors.
The invention also includes a GSK-3 inhibitor which is identified by a method comprising providing a mixture comprising GSK-3, a source of phosphate, a GSK-3 substrate and a GSK-3 assay buffer, incubating the mixture in the presence or absence of a test compound, and measuring the level of phosphorylation of the GSK-3 substrate, wherein a lower level of phosphorylation of the GSK-3 substrate in the presence of the test compound compared with the level of phosphorylation of the GSK-3 substrate in the absence of the test compound is an indication that the test compound is a GSK-3 inhibitor.
Also included in the invention is a method of treating a GSK-3 -related disorder in an animal comprising administering to the animal a GSK-3 inhibitor suspended in a pharmaceutically acceptable carrier. Preferably, the animal is a mammal, and more preferably, the mammal is a human.
The GSK-3 related disorder which is treated according to the method of the invention is preferably selected from the group consisting of bipolar disorder including mania, Alzheimer""s disease, diabetes, and leukopenia.
Lithium is recognized as a potent stimulator of hematopoiesis, both in vivo and in vitro (Doukas et al., 1986, Exp. Hematol. 14:215-221). Treatment of cyclic hematopoiesis in the grey collie dog with lithium carbonate eliminated the recurrent neutropenia and normalized the other blood cell counts (Hammond et al., 1980, Blood 55:26-28). Furthermore, lithium has been observed to stimulate in vitro Dexter culture hemopoiesis, leading to increases in granulocyte, megakaryocytes, and pluripotent stem cell numbers. In one study in a murine Dexter culture system, exposure of Dexter cultures to 1 mM LiCl prior to culture resulted in greater hemopoiesis than was observed in Dexter cultures which were not exposed to LiCl (Quesenberry et al., 1984, Blood 63:121-127). These findings suggest that human cyclic hematopoiesis, including leukopenia, may be successfully treated with lithium.
The GSK-3 inhibitor which is used to treat a GSK-3 related disorder is preferably Ro31-8220 or structurally-related compounds.
The invention also relates to a method of reducing motility of mammalian spermatozoa comprising administering to the spermatozoa a GSK-3 inhibitor suspended in a pharmaceutically-acceptable carrier. Lithium has been demonstrated to inhibit the motility of swimming spermatozoa.